RESUMO
While probing the role of RNA for the function of SET1C/COMPASS histone methyltransferase, we identified SET1RC (SET1 mRNA-associated complex), a complex that contains SET1 mRNA and Set1, Swd1, Spp1 and Shg1, four of the eight polypeptides that constitute SET1C. Characterization of SET1RC showed that SET1 mRNA binding did not require associated Swd1, Spp1 and Shg1 proteins or RNA recognition motifs present in Set1. RNA binding was not observed when Set1 protein and SET1 mRNA were derived from independent genes or when SET1 transcripts were restricted to the nucleus. Importantly, the protein-RNA interaction was sensitive to EDTA, to the translation elongation inhibitor puromycin and to the inhibition of translation initiation in prt1-1 mutants. Taken together, our results support the idea that SET1 mRNA binding was dependent on translation and that SET1RC assembled on nascent Set1 in a cotranslational manner. Moreover, we show that cellular accumulation of Set1 is limited by the availability of certain SET1C components, such as Swd1 and Swd3, and suggest that cotranslational protein interactions may exert an effect in the protection of nascent Set1 from degradation.
Assuntos
Histona-Lisina N-Metiltransferase/metabolismo , RNA Mensageiro/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Ligação a DNA/metabolismo , Ácido Edético/metabolismo , Regulação Fúngica da Expressão Gênica , Histona-Lisina N-Metiltransferase/química , Histona-Lisina N-Metiltransferase/genética , Ligação Proteica , Biossíntese de Proteínas , Estrutura Terciária de Proteína , Inibidores da Síntese de Proteínas/metabolismo , Puromicina/metabolismo , RNA Mensageiro/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genéticaRESUMO
The aim of the study was to optimize hybridization conditions of molecular probes specific for X sex chromosomes of the domestic horse in mare oocyte chromosomes. Mare oocytes, recovered from slaughterhouse ovaries by scraping the granulosa layer, were cultured in vitro. Metaphase II mature oocytes were treated with hypotonic solution and fixed, followed by hybridization of the molecular probe specific for the X chromosome ofthe domestic horse. Hybridization of probes specific for mouse heterosomes on mouse oocytes and early embryos was performed to verify the FISH technique. Of 438 oocytes analysed, 29% reached metaphase II. Despite many changes in the composition of hypotonic solutions and modification of the FISH protocol, the fluorescence signal was observed in mouse oocytes and embryos but not in mare oocytes.
Assuntos
Cavalos/genética , Hibridização in Situ Fluorescente/veterinária , Oócitos/ultraestrutura , Cromossomo X , Animais , Feminino , Hibridização in Situ Fluorescente/métodos , CamundongosRESUMO
Fluorescence in situ hybridization (FISH) is widely used in the study of chromosome structure and organization. Cytogenetic evaluation of chromosomes using FISH technique plays an increasingly important role in diagnosing karyotype changes in both somatic and reproductive cells. The aim of the study was to optimize the conditions of stallion sperm decondensation, which have a significant effect on the results of fluorescence in situ hybridization. Appropriate type and time of decondensation was chosen for the sperm of every stallion. It was found that decondensation performed using a preparation incubated in DTT solution for 1.5 minutes and in SDS solution for 10 seconds proved effective for stallions no. 1 and 2. An alternative decondensation method performed in an Eppendorf tube, with incubation in DTT solution for 1 minute and in SDS solution for 5 seconds proved effective for stallions no. 3 and 4. Decondensation using DTT and papain solution, a method successfully used for bull spermatozoa, proved inadequate for horse spermatozoa.